Application of chiral gold nanoparticles in preparation of medicine for treating Huntington's disease

Abstract

According to the method, the chiral gold nanoparticles with good biocompatibility are successfully prepared by finely controlling chemical reaction conditions. Meanwhile, a C57BL / 6 adult mouse is selected as an experimental subject, excitatory neurotoxin quinolinic acid is injected into a specific area of the brain of the mouse by adopting a stereotactic injection technology, and the Huntington's mouse model is successfully established. On the basis, the synthesized chiral gold nanoparticles are administered in an intravenous injection mode, and a PBS group and a tetrabenazine group are set as contrasts, so that the treatment effect of the chiral gold nanoparticles is evaluated. The prepared chiral gold nanoparticles can improve the structure of the cerebellum tissue of a mouse and relieve neurodegenerative diseases, a new thought and method are provided for treatment of the Huntington's disease, and the chiral gold nanoparticles have wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly relates to the application of chiral gold nanoparticles in the preparation of drugs for treating Huntington's disease. Background Art

[0002] Huntington's Disease (HD) is a hereditary neurodegenerative disease, the pathological characteristics of which are mainly the progressive death of neurons in the cerebral cortex and striatum, resulting in motor disorders, cognitive disorders and mental symptoms in patients. At present, although there are some drugs that can relieve the symptoms of HD patients, there is still no radical cure. Therefore, developing new and effective drugs for the treatment of HD is of great significance.

[0003] In recent years, the application of nanotechnology in the field of biomedicine has become increasingly widespread, providing new ideas for disease treatment. Gold nanomaterials, due to their unique physical and chemical properties and good biocompatibility, have shown great potential in drug delivery, bioimaging and disease treatment. Especially in the treatment of neurodegenerative diseases, gold nanoparticles can cross the blood-brain barrier and directly act on the lesion site to play a therapeutic role.

[0004] However, applying gold nanomaterials to the treatment of Huntington's disease still needs to overcome many challenges. For example, the survival time of HD patients after onset is about 10 to 15 years, and the effects of various drugs and control measures for various HD symptoms are not ideal. Even due to the too large drug side effects, the treatment has to be terminated. There is an urgent need for a nano-medicine with good therapeutic effect and good biocompatibility. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides the application of chiral gold nanoparticles in the preparation of drugs for treating Huntington's disease.

[0006] In one embodiment of the present invention, the administration method of the chiral gold nanoparticles is intravenous injection to ensure that the drug can quickly and effectively reach the lesion site.

[0007] In one embodiment of the present invention, the treatment cycle of the chiral gold nanoparticles is set to once a week for 4 - 8 consecutive weeks.

[0008] In one embodiment of the present invention, the administration dose of the chiral gold nanoparticles is 2 - 8 mg / kg; preferably, the administration dose is 8 mg / kg.

[0009] In one embodiment of the present invention, the drug further comprises one or more auxiliary treatment components such as a neuroprotective agent, an antioxidant or an anti-inflammatory drug to synergistically enhance the therapeutic effect of the chiral gold nanoparticles.

[0010] In one embodiment of the present invention, the pharmaceutical dosage form includes, but is not limited to, dosage forms suitable for intravenous administration such as aqueous solutions, suspensions, emulsions, etc.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] The present invention realizes the effective treatment of a mouse model of Huntington's disease, thereby significantly reducing neurodegeneration and protecting the structure and function of cerebellar tissue. CLG NPs have good biocompatibility in mice, do not cause significant negative effects on mice, and maintain sufficient treatment time, thus giving full play to their therapeutic effects. The present invention is expected to bring new treatment hopes to patients with Huntington's disease and provide new inspiration and reference for the treatment of other neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the body weight curve of mice with different treatment methods.

[0014] Figure 2 It is the stained map of mouse brain tissue sections with different treatment methods. Figure 2 a is the PBS treatment group. Figure 2 b is the CLG NPs treatment group. Figure 2 c is the tetrabenazine treatment group, scale bar: 200 μm. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0015] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments cited are not intended to limit the present invention.

[0016] Example 1

[0017] Preparation of chiral gold nanoparticles:

[0018] S1: Place 12 mL of 2.8×10 4 M HAuCl 4 and 12 mL of 0.12 M CTAC aqueous solution in a beaker. In another beaker, prepare an ice solution of 0.025 M NaBH 4 solution. Slowly add 0.5 mL of the NaBH 4 ice solution to the HAuCl 4 solution and stir at room temperature for 3 minutes until the solution turns dark brown, indicating the formation of gold nanoparticles. Age the resulting solution at 35 °C for 1.5 hours.

[0019] S2: Add 0.35 g of CTAC as a surfactant to both vials, and add 9.2 mL of deionized water to make the final concentration of CTAC 0.11 M. Place the vials in a preheated water bath at 32 °C. Add 0.012 M HAuCl 4 solution and 15 μL of 0.012 M KI solution to the two vials respectively. Add 250 μL of the reducing agent ascorbic acid solution to convert Au 3+ to Au + , and adjust the total volume of the solution to 10.5 mL. Add 60 μL of the seed solution prepared in step S1 to one vial. After shaking well, immediately take 60 μL of the solution in this vial and transfer it to the other vial, and shake well again. Let it stand for 20 minutes, then centrifuge 4 times at 3500 rpm. After removing the supernatant, redisperse the precipitate in 1.2 mL of 1.2 mM CTAB solution.

[0020] S3: Disperse the nanoparticles prepared in step S2 in 1.2 mM CTAB solution. Add 0.9 mL of 120 mM CTAB and 3.8 mL of deionized water to 0.25 mL of 12 mM chloroauric acid trihydrate to form [AuBr 4 complex. Add 0.5 mL of ascorbic acid solution to the growth solution to reduce Au 3+ to Au + . Add 6 μL of 6 mM L-GSH and 60 μL of the seed solution from step S1 to the growth solution, and the color of the solution gradually turns pink. Keep the growth solution in a water bath at 32 °C for 2.5 hours, during which the color of the solution gradually turns blue. Centrifuge the resulting solution at 13000 rpm for 6 minutes. After removing the unreacted reagents, redisperse the chiral gold nanoparticles (CLG NPs) in 1.2 mM CTAB solution and store them at 4 °C for subsequent use.

[0021] Example 2

[0022] The method for establishing a Huntington's mouse model used in the present invention is as follows: Adult C57BL / 6 mice weighing 25-30 g are selected. Sodium thiopental is administered to the mice by intraperitoneal injection at a dose of 30 mg / kg body weight. After successful anesthesia of the mice, the hair at the surgical site is shaved off, and the area is alternately disinfected three times with iodophor and 70% ethanol. 50 μL of bupivacaine is subcutaneously injected in the surgical area to reduce the pain response during the operation. Then, a scalpel is used to cut the skin on the top of the mouse's head to fully expose the parietal bone. The Bregma point and the periphery of the cranial vertex are confirmed to be on the same horizontal plane through visual inspection. According to the stereotaxic parameters of the mouse brain, the injection site is determined to be 0.3 mm in front of Bregma and 1 mm lateral, and the depth from the top of the head to the abdomen is 2.5 mm. A small hole is drilled at the predetermined injection point using a medical dental drill, and then 0.5 μL of a 300 mM excitatory neurotoxin quinolinic acid (QA) saline solution (pH = 7.4) is slowly injected through a Hamilton micro-injection needle with a diameter of 30 Gauge μL within 4 minutes. After the injection is completed, wait for 1 minute, and then slowly and smoothly withdraw the injection needle from the brain tissue. The surgical wound is sutured with a fine thread, and the surgical area is washed with saline. After that, the mice are placed back in the cage and raised normally.

[0023] The mice are randomly divided into one of the following treatment groups: (1) PBS group; (2) CLG NPs group; (3) tetrabenazine group (positive control drug) (n = 5); PBS is not treated. In the tetrabenazine group, subcutaneous injection (2 mg / kg) is performed once a week; in the CLG NPs group, synthesized chiral gold nanoparticles are injected via the tail vein, and the dosage of chiral gold nanoparticles is 2 mg / kg, 5 mg / kg, 8 mg / kg, once a week.

[0024] Example 3

[0025] Rotarod test: The experiment is carried out in an acrylic glass (300 mm × 300 mm × 400 mm) box. A copper grid with a 10 V current is placed at the bottom of the acrylic glass box. Before the experiment, the animals are placed on the rotarod for training every 5 minutes, with a rotation speed of 18 r / min for 3 consecutive days. At the start of the test, the initial rotation speed is 4 r / min, and it is increased to 40 r / min after 5 minutes. During the process of increasing the rotation speed, the time when the mice fall off the rotating shaft is recorded. Each experiment lasts for 5 minutes, and the average value of 3 experiments is calculated.

[0026] As shown in Table 1, the dropping latencies of the PBS, tetrabenazine, and low, medium, and high dose groups of CLG NPs were (43.58±8.67), (93.28±3.79), (118.74±4.37), (122.34±2.42), and (143.71±2.88) s, respectively. Compared with the model group, the dropping latency of the mice in the tetrabenazine group was significantly prolonged, indicating the effectiveness of the modeling method and the positive drug treatment; compared with the tetrabenazine group, the dropping latencies of the mice in the low, medium, and high dose groups of CLG NPs were significantly prolonged and showed a dose-dependent manner, which had a good improvement effect on the balance and coordination ability of the Huntington's disease model mice. In the present invention, the high dose group (8 mg / kg) was selected as the drug dose for subsequent experiments.

[0027] Table 1

[0028]

[0029] Example 4

[0030] Record the body weight changes of different groups continuously for 6 months to evaluate the long-term biosafety of CLG NPs chiral gold nanoparticles. Tetrabenazine is a drug clinically used for the treatment of Huntington's disease. As a positive control drug, tetrabenazine has a certain curative effect in the treatment of Huntington's disease and can provide a reference for this study. Specifically, at the end of the treatment, that is, after 6 months, the body weight change trend of the model mice (8 mg / kg) treated with chiral gold nanoparticles was basically the same as that of the tetrabenazine group, and the average body weight was significantly higher than that of the control mice treated only with PBS (as Figure 1 shown). This finding preliminarily indicates that CLG NPs did not cause significant negative effects on the physiological functions of mice in the long term, but may promote the growth of mice or maintain their better health status to a certain extent, thus indirectly proving that chiral gold nanoparticles have high biocompatibility.

[0031] Example 5

[0032] One month after treatment, the mouse brains were collected. The brain tissues were fixed with 4% paraformaldehyde, dehydrated with gradient alcohol, embedded in paraffin, cut into 4 μm sections, stained with hematoxylin-eosin (HE), dehydrated again with gradient alcohol, sealed with neutral gum, observed under an optical microscope, and the target area was selected for photographing. According to the staining results and morphological characteristics, the structure and function of the brain tissues were evaluated.

[0033] Purkinje cells are the largest neurons in the cerebellar cortex, responsible for receiving and integrating signals from other neurons, and then controlling the movement and coordination of the body. Their damage and death are the key factors leading to the progression and aggravation of symptoms of Huntington's disease. In the present invention, the morphological changes of Purkinje cells in the mouse brain tissue sections were observed by HE staining, asFigure 2 As shown by the HE staining results, there were significant differences in the morphological characteristics of the cerebellar tissues of mice in each group. In the PBS group (i.e., the control group), we observed the most severe damage to the cerebellar tissues of mice. Specifically, the number of Purkinje cells was significantly reduced, the cell arrangement was disordered, the cell nuclei were blurred, and there were obvious inflammatory cell infiltration and gliosis in the intercellular space. This indicates that without any drug treatment, the cerebellum of mice was significantly affected by the pathological process of Huntington's disease and was severely damaged in its natural state. However, in the CLG NPs group (i.e., the group treated with chiral gold nanoparticles), although the mice were injected with the excitatory neurotoxin quinolinic acid to simulate the pathological process of Huntington's disease, after treatment with CLG NPs, the degree of damage to their cerebellar tissues was significantly reduced. Specifically, the number of surviving Purkinje cells was relatively large, the cell morphology was relatively intact, and the inflammatory cell infiltration and gliosis in the intercellular space were not as severe as expected. This result preliminarily indicates that CLG NPs may have a neuroprotective effect and can alleviate the neurodegeneration in the mouse model of Huntington's disease to a certain extent. In contrast, in the tetrabenazine group (a drug clinically used to treat Huntington's disease), although we also observed a protective effect on the cerebellar tissues, its effect seemed to be slightly inferior to that of the CLG NPs group. In the cerebellar tissues of mice in the tetrabenazine group, the degree of damage to Purkinje cells was still relatively obvious, the cell arrangement was slightly disordered, and there was a certain degree of inflammatory response. In summary, CLG NPs may have more obvious advantages in improving the cerebellar tissue structure of mice and alleviating neurodegeneration. This suggests that CLG NPs may become a new and effective treatment strategy for Huntington's disease.

[0034] The embodiments described above are only a part of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but only represents the selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

Claims

1. Application of chiral gold nanoparticles in the preparation of drugs for treating Huntington's disease.

2. The use according to claim 1, characterized in that: The chiral gold nanoparticles are administered by intravenous injection.

3. The use according to claim 1, characterized in that: The injection cycle of the chiral gold nanoparticles is set to be once a week, and the administration is continued for 4 to 8 weeks.

4. The use according to claim 1, characterized in that: The dosage of the chiral gold nanoparticles is 2-8 mg / kg.

5. The use according to claim 1, characterized in that: The preferred dosage of the chiral gold nanoparticles is 8 mg / kg.

6. The use according to claim 1, characterized in that: The medicament may also contain one or more auxiliary therapeutic ingredients among neuroprotectants, antioxidants or anti-inflammatory drugs.

7. The use according to claim 1, characterized in that: The pharmaceutical dosage forms include, but are not limited to, aqueous solutions, suspensions, emulsions and other dosage forms suitable for intravenous administration.

8. The use according to claim 1, characterized in that: The chiral gold nanoparticles are biocompatible, have an improving effect on the balance and coordination ability of Huntington's disease model mice, and can improve the cerebellar tissue structure of Huntington's disease model mice and alleviate neurodegenerative lesions.

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